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| Status |
Public on Mar 31, 2023 |
| Title |
200385-mNG-mAID_Veh._REP2 |
| Sample type |
SRA |
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| Source name |
intracellular protozoan parasite, cultured in HFFs
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| Organism |
Toxoplasma gondii |
| Characteristics |
strain: ME49
cell type: intracellular protozoan parasite, cultured in HFFs
genotype: ME49/TIR1/200385-mNG-mAID
condition: 96hr alkaline stress
treatment: Vehicle (PBS)
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| Treatment protocol |
To establish infection, parasites were mechanically released from host cells by passage through a 27-gauge needle and innoculated onto confluent human foreskin fibroblasts in standard medium.
After 4h, infected monolayers were washed twice with PBS, switched to alkaline-stress medium (RPMI 1640 supplemented with 1% IFS, 10 μg/mL gentamicin, 50 mM HEPES, and pH-adjusted to 8.1 with 10 N NaOH), and treated with either 5uM 3-indole-acetic acid (IAA) or an equivalent volume of vehicle.
Cultures were maintained under their respective treatment condition for 96h before harvest, with fresh stress medium provided on day 2.
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| Growth protocol |
For routine maintenance, parasites were cultured in human foreskin fibroblasts (HFFs) grown at 37°C under 5% CO2 in standard medium, consisting of DMEM (GIBCO) supplemented with 3% heat-inactivated fetal bovine serum (IFS), 2 mM L-glutamine, and 10 μg/mL gentamicin.
Passaging was performed on a 2-3 day cycle—prior to lysis of host cell monolayers—by scraping and transfer to fresh HFFs.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Parasites were isolated from host cells by syringe-release (described above) and filtering through a Millex filter unit (5 μm pore size, Merck Millipore) to remove host cell debris. RNA was isolated using the Rneasy mini plus kit (Qiagen).
Libraries for RNA-seq were prepared using the Swift Low-Input Library Prep Kit with poly(A) enrichment following manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were trimmed to remove adapter sequences using Cutadapt v.3.6
Alignment to the T. gondii genome was performed with STAR v.2.7.0a.
Differential expression analysis was done using the DESeq2 package (v. 1.12.3) in R
Assembly: T. gondii ME49 (ToxoDB v.56 assembly)
Supplementary files format and content: The corresponding xls file includes the following processed data, annotated as indicated in brackets:
Supplementary files format and content: i) log2(fold-change) in mRNA abundance in parasites depleted for each factor (i.e. treated with IAA) as compared to the IAA-treated parental TIR1 strain [L2FC.TargetedGeneID_IAA.v.TIR1_IAA]
Supplementary files format and content: ii) negative log-transformed p-values for differential expression analysis [nL10p.TargetedGeneID_IAA.v.TIR1_IAA]
Supplementary files format and content: iii) normalized transcript levels in each knockdown strain, expressed as log-transformed Fragments per Kilobase Mapped [L10FPKM.TargetedGeneID_IAA]
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| Submission date |
Jan 26, 2023 |
| Last update date |
Mar 31, 2023 |
| Contact name |
Sebastian Lourido |
| E-mail(s) |
[email protected]
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| Phone |
617-324-4920
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| Organization name |
Whitehead Institute
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| Lab |
Lourido
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| Street address |
455 Main Street, Rm 555
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL26742 |
| Series (1) |
| GSE223819 |
Effect of depleting BFD1-regulated gene-expression factors (TGME49_253790, TGME49_208020, TGME49_224630, TGME49_306620, TGME49_311100) on the Toxoplasma transcriptome during alkaline stress |
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| Relations |
| BioSample |
SAMN32934233 |
| SRA |
SRX19193677 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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